The molecular basis for the ability of host cells to avoid activation of autologous complement on their surfaces has been the subject of extensive investigations. Although there is evidence that multiple mechanisms may be involved, recent studies indicate that a 70 kilodalton membrane component, originally characterized as "decay accelerating factor" (DAF) of preformed C3 convertases, plays an important role in this protection. We have found, that DAF, in fact, blocks the uptake of C2 and factor B by C4b and C3b fragments that deposit on host cell surfaces and thereby functions primarily to prevent the assembly of C3 and C5 convertases rather than to regulate these enzymes. Recent evidence indicates that DAF is deficient in erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH) and suggests that this deficiency is causally related to the sensitivity of the cells to complement-mediated lysis in vivo. We have purified DAF to homogeneity and prepared monoclonal anti-DAF antibodies. Using these reagents, we have developed methods for the quantitation of endogenous DAF in cells, for the incorporation of exogenous DAF into cell membranes, and for the quantitative analysis of DAF function at various stages of the complement cascade. We now propose to further investigate the function of DAF in normal cells, to define the role of diminished DAF content of PNH cells in disease pathogenesis, and to try to restore the function of the PNH cells in vitro. Specifically, we propose to 1) precisely characterize normal and PNH blood cells with respect to DAF expression, 2) determine whether DAF acts at additional steps of the cascade and whether incorporation of DAF in PNH cells partially or completely restores the various lytic defects of the cells, 3) search for DAF polymorphisms to study the genetic control of DAF expression, 4) examine how phenotypic variations in C4, particularly those which lead to differences in covalent asociation with cell surface acceptor groups, affect DAF function, and 5) determine the precise nature of the DAF ligand(s) and further clarify the mechanism of DAF action.